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Regulation of Ribosomal DNA Amplification by the TOR Pathway

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Regulation of Ribosomal DNA Amplification by the TOR Pathway Regulation of ribosomal DNA amplification by the TOR pathway Carmen V. Jacka,1, Cristina Cruza,1, Ryan M. Hulla, Markus A. Kellerb, Markus Ralserb,c, and Jonathan Houseleya,2 aEpigenetics Programme, The Babraham Institute, Cambridge CB22 3AT, United Kingdom; bCambridge Systems Biology Centre and Department of Biochemistry, University of Cambridge, Cambridge CB2 1GA, United Kingdom; and cDivision of Physiology and Metabolism, Medical Research Council National Institute for Medical Research, London NW7 1AA, United Kingdom Edited by Jasper Rine, University of California, Berkeley, CA, and approved June 26, 2015 (received for review March 27, 2015) Repeated regions are widespread in eukaryotic genomes, and key The rate of recombination between copies is regulated by the functional elements such as the ribosomal DNA tend to be formed histone deacetylase (HDAC) Sir2 (11-13), and recombination of high copy repeated sequences organized in tandem arrays. In through this pathway is strictly dependent on the homologous general, high copy repeats are remarkably stable, but a number of recombination (HR) machinery (14). Frequent recombination organisms display rapid ribosomal DNA amplification at specific events are required to maintain rDNA homogeneity (15, 16) and times or under specific conditions. Here we demonstrate that result in the loss of markers integrated in the rDNA (7). However, target of rapamycin (TOR) signaling stimulates ribosomal DNA this HR-dependent pathway regulated by Sir2 is nondirectional; amplification in budding yeast, linking external nutrient avail- repeat gain and loss occurs at equivalent rates, so no change in ability to ribosomal DNA copy number. We show that ribosomal average copy number is observed over time (13). DNA amplification is regulated by three histone deacetylases: Sir2, Nonetheless, concerted increases in rDNA copy number occur Hst3, and Hst4. These enzymes control homologous recombina- in Saccharomyces cerevisiae populations with low or limiting tion-dependent and nonhomologous recombination-dependent rDNA copy number (10, 17), as well as in a variety of other amplification pathways that act in concert to mediate rapid, organisms (18, 19), showing that an rDNA amplification pathway directional ribosomal DNA copy number change. Amplification is must also exist. This may overlap with the Sir2-regulated path- completely repressed by rapamycin, an inhibitor of the nutrient- way but must be distinct, as complete de-regulation of rDNA responsive TOR pathway; this effect is separable from growth rate BIR in sir2Δ mutants does not cause constitutive rDNA ampli- and is mediated directly through Sir2, Hst3, and Hst4. Caloric fication (13). In contrast, constitutive rDNA amplification has restriction is known to up-regulate expression of nicotinamidase been reported in cells lacking histone H3 K56 acetyltransferase Pnc1, an enzyme that enhances Sir2, Hst3, and Hst4 activity. In activity, suggesting H3 K56 acetylation may control entry to an contrast, normal glucose concentrations stretch the ribosome rDNA amplification pathway (20, 21). Surprisingly, rDNA am- synthesis capacity of cells with low ribosomal DNA copy number, and we find that these cells show a previously unrecognized plification can occur in the absence of critical HR proteins, in- transcriptional response to caloric excess by reducing PNC1 expres- cluding strand exchange factor Rad52 (20), suggesting two sion. PNC1 down-regulation forms a key element in the control of mechanistically separable pathways exist: the HR-dependent ribosomal DNA amplification as overexpression of PNC1 substan- BIR pathway and a non-HR-dependent amplification pathway. tially reduces ribosomal DNA amplification rate. Our results reveal Advantageous copy number changes are generally assumed to how a signaling pathway can orchestrate specific genome changes occur at random and then spread through a population by natural and demonstrate that the copy number of repetitive DNA can be selection. However, increasing rDNA copy number in yeast does altered to suit environmental conditions. not provide a detectable growth advantage under laboratory ribosomal DNA | homologous recombination | Sir2 | Significance copy number variation | TOR We tend to think of our genome as an unchanging store of in- ukaryotic genomes contain abundant multicopy sequences, formation; however, recent evidence suggests that genomes Eranging from low copy segmental duplications to the giant vary between different cells in the same organism. How these tandem arrays found at key functional regions such as centro- differences arise and what effects they have remain unknown, meres, telomeres, and the ribosomal DNA (rDNA) (1). Copy but clearly our genome can change. In a single-celled organism, number variation of protein coding genes has been linked with genome changes occur at random, and advantageous changes multiple diseases, suggesting copy number has significant effects slowly propagate by natural selection. However, it is known that on gene expression (2, 3). The budding yeast rDNA has been the DNA encoding ribosomes can change simultaneously in a used extensively as a model system for dynamic copy number whole population. Here we show that signaling pathways that change in repetitive DNA. The rDNA consists of a tandem array sense environmental nutrients control genome change at the ribosomal DNA. This demonstrates that not all genome changes of ∼180 tandem copies, each containing genes for the 35S and 5S occur at random and that cells possess specific mechanisms to preribosomal RNAs. rDNA copy number is stable in a pop- optimize their genome in response to the environment. ulation, but recombination between rDNA copies is frequent because of the presence of a recombination-stimulating HOT1 Author contributions: C.V.J., C.C., M.A.K., M.R., and J.H. designed research; C.V.J., C.C., element in each copy (4–7). The HOT1 element includes a R.M.H., and M.A.K. performed research; C.V.J., C.C., R.M.H., M.A.K., and J.H. analyzed unidirectional replication fork barrier dependent on the Fob1 data; and J.H. wrote the paper. protein that halts replication forks moving in the opposite di- The authors declare no conflict of interest. rection to RNA Pol I (8); Fob1 is required both for ectopic This article is a PNAS Direct Submission. HOT1 activity and for rDNA recombination (9). Freely available online through the PNAS open access option. The primary model for Fob1-stimulated recombination involves 1C.V.J. and C.C. contributed equally to this work. breakage of a replication fork stalled at the replication fork bar- 2To whom correspondence should be addressed. Email: [email protected]. rier, leaving a single-ended double-strand break that can initiate This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. break-induced replication (BIR) with the sister chromatid (10). 1073/pnas.1505015112/-/DCSupplemental. 9674–9679 | PNAS | August 4, 2015 | vol. 112 | no. 31 www.pnas.org/cgi/doi/10.1073/pnas.1505015112 conditions, so rapid rDNA amplifications cannot be explained by such a mechanism (10, 22). This implies the existence of a mech- anism that can monitor rDNA copy number and instigate rDNA amplification when required. The target of rapamycin (TOR) pathway stimulates marker loss from the rDNA via the nondirectional BIR pathway (23, 24) and is also known to mod- ulate H3 K56 acetylation in the rDNA (25). Because the TOR pathway responds to environmental nutrient availability (26) and represses rDNA recombination during caloric restriction (24, 27), we asked whether TOR signaling controls rDNA amplification. Here we show that rDNA amplification in budding yeast occurs through two pathways that are coordinately regulated by TOR signaling, providing a clear demonstration that the copy number of certain loci can be tailored to suit the current environment. Results rDNA Amplification Is Controlled by the TOR Pathway. rDNA copy number is stably maintained at 150–200 repeats in wild-type yeast, and cells with low rDNA copy numbers (fewer than ∼80 copies) undergo rapid amplification toward the wild-type level (10, 17). However, low copy number rDNA arrays cannot am- plify in the absence of Fob1, and amplification in fob1Δ cells is initiated by the introduction of a Fob1 expression plasmid (17, 22, 28). We exploited this assay to test whether TOR signaling is required for rDNA amplification in cells with ∼35 rDNA repeats (rDNA35), which, in accord with previous data, have only a minimal growth defect compared with isogenic cells with 180 GENETICS rDNA copies (SI Appendix, Fig. S1 A and B). The rDNA array occupies ∼40% of chromosome XII in wild- type yeast, and the migration of chromosome XII by pulsed-field gel electrophoresis (PFGE) is routinely used to assay rDNA copy number. Heterogeneous chromosome XII signals indicate rDNA copy number heterogeneity in the population (e.g., Fig. 1A, compare lanes 1 and 2). Other chromosomes are shown by ethidium staining to control for loading and genome stability. Fig. 1. The TOR pathway controls rDNA amplification. (A) rDNA35 cells in Multiple clones are routinely tested, and the PFGE data can be which FOB1 is deleted (lane 1) were transformed with a pFOB1 plasmid that combined into average rDNA copy number distribution plots expresses FOB1 from the endogenous promoter. Half of the transformation (e.g., Fig. 1A, Upper right, derived from Fig. 1A, Left, lanes 1–7). mix was plated without rapamycin (lanes 2–4), and half with rapamycin – rDNA35 cells, which lack the FOB1 gene, were transformed (lanes 5 7), with three colonies from each transformation analyzed after with a plasmid expressing FOB1 from the endogenous promoter three restreakings (∼60 generations). Cells from lanes 5–7 were restreaked (pFOB1), and multiple transformants were grown in the pres- four times without rapamycin (lanes 8–10) or with rapamycin (lanes 11–13). Cells were grown to stationary phase in liquid culture with or without ence or absence of the TOR inhibitor rapamycin. rDNA35 cells underwent rapid rDNA amplification on introduction of the FOB1 rapamycin, they were lysed, and chromosomes were separated by PFGE.
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